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Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular level. We have shown earlier that active repression of telomerase gene expression by chromatin remodelling of the promoters is one mechanism of regulation; however, other genes and signalling networks are likely to be required to regulate telomerase and maintain the ALT phenotype. Using gene expression profiling, we have uncovered a signature of 1305 genes to distinguish telomerase-positive and ALT cell lines. By combining this with the gene expression profiles of liposarcoma tissue samples, we refined this signature to 297 genes. A network analysis of known interactions between genes within this signature revealed a regulatory signalling network consistent with a model of human telomerase reverse transcriptase (hTERT) repression in ALT cell lines and liposarcomas. This network expands on our existing knowledge of hTERT regulation and provides a platform to understand differential regulation of hTERT in different tumour types and normal tissues. We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues.

Telomere length is maintained by two known mechanisms, activation of telomerase or alternative lengthening of telomeres (ALT). The ALT pathway is more commonly activated in tumours of mesenchymal origin, although the mechanisms involved in the decision of a cell to activate either telomerase or ALT are unknown at present and no molecular markers exist to define the ALT phenotype. We have previously shown an association between chromatin remodelling, telomerase gene expression and ALT in cell line models. Here, we evaluate these findings and investigate their prognostic significance in a panel of liposarcoma tissue samples to understand the biology underlying the ALT phenotype. Liposarcoma samples were split into three groups: telomerase positive (Tel+); ALT positive; ALT-/Tel-. Differences in telomerase gene expression were evident between the groups with increased expression of hTR in ALT and Tel+ compared to ALT-/Tel- samples and increased hTERT in Tel+ samples only. Investigation of a small panel of chromatin modifications revealed significantly increased binding of acetyl H3 in association with hTR expression. We confirm that the presence of the ALT phenotype is associated with poor prognosis and in addition, for the first time, we show a direct association between hTR expression and poor prognosis in liposarcoma patients.

Objectives: Peptide nucleic acids (PNAs) are DNA mimics that have been demonstrated to be efficient antisense/antigene tools in cell-free systems. However, their potential as in vivo regulators of gene expression has been hampered by their poor uptake by living cells, and strategies need to be developed for their intracellular delivery. This study has aimed to demonstrate the possibility (i) of efficiently delivering a PNA, which targets mRNA of the catalytic component of human telomerase reverse transcriptase (hTERT), into DU145 prostate cancer cells through a combined approach based on conjugation of the PNA to Tat internalizing peptide (hTERT-PNA-Tat) and subsequent photochemical internalization, and (ii) to interfere with telomerase function., Materials and Methods: Treated cells were analysed for telomerase activity, hTERT expression, growth rate, ability to undergo apoptosis and telomere status., Results: After exposure to light, DU145 cells treated with hTERT-PNA-Tat and the photosensitiser TPPS2a showed dose-dependent inhibition of telomerase activity, which was accompanied by marked reduction of hTERT protein expression. A dose-dependent decline in DU145 cell population growth and induction of caspase-dependent apoptosis were also observed from 48 h after treatment. Such an antiproliferative effect was associated with the presence of telomeric dysfunction, as revealed by cytogenetic analysis, in the absence of telomere shrinkage, and with induction of DNA damage response as suggested by the increased expression of gamma-H2AX., Conclusions: Our results (i) indicate photochemical internalization as an efficient approach for intracellular delivery of chimaeric PNAs, and (ii) corroborate earlier evidence suggesting pro-survival and anti-apoptotic roles of hTERT, which are independent of its ability to maintain telomere length.

The expression of telomerase in human cells is strictly controlled by multiple mechanisms including transcription and alternative splicing of telomerase reverse transcriptase (hTERT). In this study, we demonstrated the possibility of modulating the hTERT splicing pattern in DU145 human prostate carcinoma cells through the use of 2'-O-methyl-RNA phosphorothioate oligonucleotides targeting the splicing site located between intron 5 and exon 6 in the hTERT pre-mRNA. An 18-h oligonucleotide exposure induced a decrease in the full-length hTERT transcript and a concomitant increase in the alternatively spliced transcripts, which resulted in significant inhibition of telomerase catalytic activity. Moreover, exposure to the R7 oligomer (which induced the most pronounced modulation of the hTERT splicing pattern and the greatest telomerase inhibition) caused a marked reduction in DU145 cell growth and the induction of apoptosis starting 2 days after treatment. Such data support the concept that down-regulation of hTERT expression can cause short-term effects on tumour cell growth, which are telomere-shortening independent.

Telomerase activity was determined in 15 breast cancers, 24 benign breast lesions, and 36 breast tissues adjacent to benign or malignant tumours. A positive TRAP (telomeric repeat amplification protocol) signal was detected in 67% of carcinomas and 29% of benign tumours. In five of ten cases, non-invaded breast tissues adjacent to telomerase-positive carcinomas also displayed telomerase activity. Conversely, in peritumoural specimens adjacent to benign lesions, telomerase activity was never detected. To investigate the regulatory mechanisms of telomerase activity in breast tissues, the expression of telomerase subunits was assessed, as well as the presence of alternatively spliced variants of human telomerase reverse transcriptase (hTERT). The presence of the hTERT full-length transcript appeared necessary for telomerase activity in breast carcinomas. Specifically, all telomerase-positive carcinomas expressed the hTERT full-length message, together with different combinations of alternatively spliced variants, whereas in telomerase-negative cancers, the hTERT full-length transcript was not detectable, or its abundance was markedly lower than that of alternatively spliced variants. Results obtained in benign tumours and normal tissues surrounding carcinomas instead showed that the presence of hTERT full-length transcript was not sufficient to determine telomerase activity. These findings suggest that in non-neoplastic tissues there are other mechanisms that suppress telomerase activity downstream from hTERT transcription and mRNA splicing and that such mechanisms have been lost during neoplastic transformation., (Copyright 2002 John Wiley & Sons, Ltd.)

In this study, we evaluated the potential of the distamycin derivative MEN 10716 as a telomerase inhibitor. Exposure of human melanoma cell extracts to MEN 10716 induced a dose-dependent inhibition of telomerase activity, with an IC50 of 24+/-3 microM. When intact JR8 melanoma cells were chronically exposed to the drug (200 microM every other day for 50 days), a marked inhibition (>80%) of the enzyme's catalytic activity was consistently observed starting from day 1. At later points in time, MEN 10716 inhibited melanoma cell proliferation and induced apoptosis. Cells surviving MEN 10716 exposure were characterised by a higher melanin content and a greater expression of p16(INK4A) protein than control cells. The effects of MEN 10716 were subsequently evaluated in different tumour cell systems. In particular, even in the H460 non-small cell lung cancer cell line, chronic exposure of the cells to the drug (100 microM every other day for 50 days) induced a consistent inhibition (>85%) of telomerase activity, a reduction of cell proliferation potential, and apoptosis. Conversely, MEN 10716 treatment did not appreciably inhibit cell proliferation in the U2-OS telomerase-negative human osteogenic sarcoma cell line. Interestingly, no variation in the mean telomere length was observed in MEN 10716-treated JR8 melanoma cells, whereas an appreciable increase in the mean telomere length was found in H460 lung cancer cells after drug exposure. Overall, the results of the study indicate that MEN 10716 is a possible telomerase inhibitor and suggest that abrogation of telomerase activity can affect cell proliferation even through pathways that are not dependent on telomere erosion.

To investigate the regulatory mechanisms of telomerase activity in human melanoma cells, we assessed the enzyme's catalytic activity and the expression of the telomerase subunits, the human telomerase RNA, the human telomerase-associated protein, and the human telomerase reverse transcriptase, in 52 melanoma lesions. Eight normal skin specimens were also studied. Telomerase activity was detected in 84.6% of melanomas, whereas all skin specimens were telomerase negative. Human telomerase-associated protein mRNA and human telomerase RNA were constitutively expressed in all melanoma and skin specimens. Although at a variable level of expression, human telomerase reverse transcriptase mRNA was detected in all but one melanomas, whereas it was never present in skin samples. Reverse transcriptase-polymerase chain reaction experiments were performed using primers within the reverse transcriptase domain of human telomerase reverse transcriptase and revealed the presence of multiple alternatively spliced transcripts in melanoma specimens. Among the 44 telomerase-positive melanomas, one showed the full-length transcript alone whereas in all other specimens a full-length message was present with different combinations of alternatively spliced variants. In these tumors the expression of the full-length transcript was generally equal to or higher than that of the alternatively spliced variants. The ratio full-length transcript to alternatively spliced species ranged from 0.6 to 5.26, with a median value of 1.18. Among the seven telomerase-negative melanomas, one displayed the beta deletion transcript alone, whereas in the remaining six tumors weak expression of the full-length transcript and a more abundant level of alternatively spliced transcripts were found. In these cases human telomerase reverse transcriptase ratio ranged from 0.09 to 1.1, with a median value of 0.40. The results suggest that transcription and alternative splicing of human telomerase reverse transcriptase are regulatory mechanisms controlling telomerase activity in melanoma.

In tumour cells, replicative immortality is attained through stabilisation of telomeres by telomerase. Recent evidence suggests that telomerase plays an anti-apoptotic role. Since apoptosis is the primary mode of cell death induced by several drugs, telomerase could be involved in determining the chemosensitivity profile of tumour cells. We investigated whether inhibition of telomerase activity through a hammerhead ribozyme targeting the RNA template of telomerase influences the susceptibility of human melanoma cells to a variety of anticancer agents (platinum compounds, taxanes, topoisomerase I inhibitors). The ribozyme sequence was inserted into an expression vector and the JR8 human melanoma cell line was transfected with it. The cell clones obtained showed a reduced telomerase activity. Growth inhibition curves generated after exposure of ribozyme-transfectant clones to individual drugs were superimposable to those obtained from parental cells. Moreover, telomerase inhibition did not promote apoptosis as a cellular response to drug treatment. Overall, our results indicate that downregulation of telomerase activity does not increase the sensitivity of melanoma cells to anticancer drugs.

We investigated the effect of two peptide nucleic acids (PNAs), which are complementary to the RNA component of human telomerase, on the catalytic activity of the enzyme. PNAs induced a dose-dependent reduction of telomerase activity in cell extracts from human melanoma cell lines and surgical specimens. To down-regulate telomerase in intact cells, we generated a chimeric molecule synthesized by coupling the 13-mer PNA to the Antennapedia peptide. The PNA construct induced a dose- and time-dependent inhibition of telomerase activity. However, a 20-day exposure to the PNA construct only caused a slight increase in melanoma cell doubling time and failed to induce any telomere shortening.

Since telomerase plays a role in cellular resistance to apoptosis, which is the primary mode of cell death induced by several drugs, telomerase could be involved in determining the chemosensitivity profile of tumor cells. Thus, we investigated the relationship between telomerase activity, telomere length and chemosensitivity to effective antitumor agents in a panel of human melanoma and ovarian cancer cell lines. Telomerase activity, as detected by the telomeric repeat amplification protocol, ranged from 0.58 to 1.10 arbitrary units in individual cell lines, with similar median values for melanoma and ovarian carcinoma cell lines (0.80 vs. 0.90). Telomeres were generally longer in melanoma than in ovarian carcinoma cell lines, with a more than 2-fold median telomere restriction fragment length (7.74 vs. 3.82 kb). No significant correlation was evidenced between the two telomere-related parameters and cell population doubling time, DNA index or TP53 gene status. No precise relation was found between telomerase activity and cellular sensitivity to different DNA damaging agents including doxorubicin, cisplatin and the multinuclear platinum compound BBR 3464. In contrast, longer telomeres were associated to resistance to the drugs, even though the association reached statistical significance only for cisplatin. Since platinum compounds may have affinity for telomere sequences, it is conceivable that the interaction is relevant for drug sensitivity/resistance status depending on telomere length.

Reactivation of telomerase, an RNA-dependent DNA polymerase that synthesizes new telomeric repeats at the end of chromosomes, is a very common feature in human cancers. Telomerase is thought to be essential in maintaining the proliferative capacity of tumor cells and, as a consequence, it could represent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanking sequences complementary to the RNA component of human telomerase and designed to cleave specifically a site located at the end of the telomerase template sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts from two human melanoma cell lines (JR8 and M14), or three melanoma surgical specimens, the ribozyme inhibited telomerase activity in a concentration- and time-dependent manner. When the ribozyme was delivered to growing JR8 melanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate-mediated transfer, a marked inhibition of telomerase activity was observed. Next, the ribozyme sequence was cloned in an expression vector and JR8 cells were transfected with it. The cell clones obtained showed a reduced telomerase activity and telomerase RNA levels and expressed the ribozyme. Moreover, ribozyme transfectants had significantly longer doubling times than control cells and showed a dendritic appearance in monolayer culture. No telomere shortening, however, was observed in these clones. Overall, our results indicate that the hammerhead ribozyme is a potentially useful tool for the inactivation of telomerase in human tumors.

Focuses on the enzyme telomerase which medical researchers believe may contribute to tumorigenesis and neoplastic progression. Effort to determine at what stage of cancer development telomerase in reactivated; Why scientists have focused their efforts on defining the utility of assaying for telomerase as a diagnostic tool.